Xylanase, microorganisms producing it, DNA molecules, methods for preparing this xylanase and uses of the latter

ABSTRACT

The invention relates to a xylanase originating from a  Bacillus  strain. This xylanase is active over a wide range of acid and basic pH. 
     The invention also relates to new strains of microorganisms producing this xylanase and to methods for preparing this xylanase. 
     The invention also relates to a DNA molecule and to an expression vector or an integration vector containing this DNA molecule. 
     The invention also relates to uses of the latter and to compositions containing it.

This is a divisional of application Ser. No. 09/909,207 filed Jul. 19,2001 now U.S. Pat. No. 7,022,827.

The invention relates to a new xylanase. The invention also relates tothe methods for preparing this xylanase, to the uses of the latter andto compositions comprising it.

The invention also relates to a new strain of microorganisms producingthis xylanase and to a DNA molecule comprising the nucleotide sequencewhich codes for this xylanase. The invention also relates to vectorscontaining this DNA molecule and to strains transformed by thesevectors.

The invention also relates to the promoter derived from the gene whichcodes for Bacillus pumilus PRL B12 xylanase and the presequence whichcodes for the signal peptide of Bacillus pumilus PRL B12 xylanase. Theinvention also relates to vectors which contain this promoter and thispresequence, and also to the DNA molecule comprising the nucleotidesequence which codes for the mature portion of the xylanase of theinvention. The invention also relates to strains transformed by thesevectors.

Thermostable xyalanases which are active over a wide pH range are known,such as, in particular, xylanases produced by strains of alkalophilicbacillus (Gupta et al. Biotechnology Letters, 1992, 14 (11), pages1045-1046 and International Patent Application WO 94/04664). However,despite these properties, these enzymes would appear to be poorlyeffective in bleaching paper pulp.

Consequently, there is at present a need for a xylanase which can beused in the treatment of paper pulp, which is very stable and also veryactive over a wide range of temperature and of basic and acid pH.

The object of the present invention is to provide a new xylanase whichis active over a wide pH range, both at alkaline pH and at acid pH.

The object of the present invention is also to identify, isolate andprovide a strain, especially a Bacillus strain, which produces the saidxylanase naturally.

The object of the present invention is also to isolate and provide a DNAmolecule comprising a nucleotide sequence which codes for the saidxylanase.

The object of the present invention is also to prepare and provide anexpression vector containing the nucleotide sequence coding for the saidxylanase.

The object of the present invention is also to prepare and provide anintegration vector containing the nucleotide sequence coding for thesaid xylanase.

The object of the present invention is also to prepare and provide thepromoter drived from the gene which codes for Bacillus pumilus PRL B12xylanase. The object of the present invention is also to prepare andprovide the presequence which codes for the signal peptide of Bacilluspumilus PRL B12 xylanase. The vectors which comprise this promoterand/or this presequence also contain the DNA molecule comprising thenucleotide sequence which codes for the mature portion of the xylanaseof the invention. The strains transformed by these vectors produce thexylanase of the invention heterologously.

The object of the present invention is also to prepare and provide aBacillus host transformed with the expression vector which contains theDNA molecule comprising the nucleotide sequence of, the Bacillus straincoding for the said xylanase.

The object of the present invention is also to prepare and provide aBacillus host transformed with the expression vector which contains theDNA molecule comprising the nucleotide sequence of the Bacillus straincoding for the said xylanase [sic].

The object of the present invention is also to prepare and provide acomposition containing this xylanase.

The object of the present invention is also to prepare and provide axylanase which can be used in the treatment of paper pulp, and pulpshaving a basic, neutral or acid pH, and in particular pulps having anespecially basic pH and paper pulps of various origins, such as thepulps originating from coniferous trees, the pulps originating frombroad-leaved trees and especially eucalyptus pulp.

To this end, the invention relates to a xylanase originating from aBacillus, and more especially from an aerobic and non-thermophilicmicroorganism.

It is preferable to use Bacillus sp. strain 720/1 or a derivative ormutant of this strain. The xylanase of the invention is derived from(naturally produced by) Bacillus sp. strain 720/1. Xylanase isclassified in the international system under the EC number 3.2.1.8. Itis an endo-1,4-beta-xylanase.

Preferably, the isolated and purified xylanase consists of a single typeof polypeptide having a molecular weight of approximately 25 kDa.

The invention relates to an isolated and purified xylanase comprisingthe amino acid sequence from 1 to 221 amino acids (SEQ ID NO:3) or amodified sequence derived from this sequence. The amino acid sequenceand the nucleotide sequence (SEQ ID NO:1) coding for the maturexylanase, together with its translation into amino acids (SEQ ID NO:2),is given in FIG. 1 (FIGS. 1 a and 1 b).

The xylanase of the invention is synthesized in the form of a precursor.The precursor contains 248 amino acids: (SEQ ID NO:6). The nucleotidesequence SEQ ID NO:4) coding for the xylanase precursor, as well as itstranslation into amino acids (SEQ ID NO:5), are identified.

The precursor contains the sequence of 221 amino acids (SEQ ID NO:3) ofthe mature xylanase and the sequence of 27 amino acids (SEQ ID NO:9) ofthe presequence.

The mature xylanase sequence is preceded by a presequence. The latter isan additional sequence of 27 amino acids (SEQ ID NO:9). Thecorresponding nucleotide sequence (SEQ ID NO:7), as well as itstranslation into amino acids (SEQ ID NO:8), are identified. Thispresequence codes for the signal peptide of the xylanase of theinvention.

As a special preference, the said xylanase has a determined isoelectricpoint of between approximately 9.5 and approximately 9.7.

The xylanase according to the invention is thermostable and active overa wide pH range. Preferably, the xylanase according to the invention isalkaline.

The xylanase according to the invention possesses, moreover, allappropriate properties compatible with the actual industrial conditionsof enzyme treatment of paper pulp. According to the numerous steps ofthe various treatments of paper pulp employed industrially, theseproperties are good stability with respect to pH and temperature, andenzyme activity over a wide range of pH and temperature, such as, inparticular, a pH of between approximately 5 and 10 and a temperature ofbetween approximately 50 and 80° C.

The xylanase of the invention is active over a range of pH above orequal to approximately 5. The xylanase of the invention is active over arange of pH lower or equal to approximately 11. The xylanase develops anenzyme activity of more than 50% of the maximal activity, measured at atemperature of approximately 50° C. and in the presence of xylan, over arange of pH above or equal to approximately 5.0. The xylanase developsan enzyme activity of more than 50% of the maximal activity, measured ata temperature of approximately 50° C. and in the presence of xylan, overa pH range below approximately 10.5.

The xylanase of the invention is active over a range of temperatureabove or equal to approximately 50° C. The xylanase of the invention isactive over a range of temperature below or equal to approximately 80°C. The xylanase develops an enzyme activity of more than 50% of themaximal activity, measured at a pH of approximately 9 and in thepresence of xylan, over a range of temperature above or equal toapproximately 50° C. The xylanase develops an enzyme activity of morethan 50% of the maximal activity, measured at a pH of approximately 9and in the presence of xylan, over a temperature range belowapproximately 80° C.

The invention also relates to a modified xylanase, that is to say anenzyme whose amino acid sequence differs from that of the wild-typeenzyme by at least one amino acid. These modifications may be obtainedby standard mutagenesis techniques on the DNA, such as exposure toultraviolet radiation or to chemical products such as ethylmethanesulphonate (EMS), N-methyl-N-nitro-N-nitrosoguanidine (MNNG),sodium nitrite or O-methylhydroxylamine, or by genetic engineeringtechniques such as, for example, site-directed mutagenesis or randommutagenesis. These techniques are known to a person skilled in the artand are described, in particular, in Molecular Cloning—a laboratorymanual—Sambrook, Fritsch, Maniatis—second edition, 1989, Chapter 15.

The invention also relates to a xylanase having immunochemicalproperties identical or partially identical to the xylanase obtainedfrom Bacillus sp. strain 720/1. The immunochemical properties may bedetermined immunologically by tests of identity, in particular usingspecific polyclonal or monoclonal antibodies. Tests of identity areknown to a person skilled in the art, such as, in particular, theOuchterlony immunodiffusion method or the immunoelectrophoresis method.Examples of such methods are described by Axelsen N. H., Handbook ofImmunoprecipitation Gel Techniques, Blackwell Scientific Publications,1983, Chapters 5 and 14; the terms “antigenic identity” and “partialantigenic identity” are described in this document in Chapters 5, 19 and20. A serum containing the specific antibody is prepared according tothe method described, by immunizing animals (for example mice, rabbitsor goats) with a purified xylanase preparation. This preparation may bemixed with an additive such as Freund's adjuvant, and the mixtureobtained is injected into animals. The polyclonal antibody is obtainedafter one or several immunizations. An example consists in injectingsubcutaneously at two-week intervals four fractions each containing 150micrograms of purified xylanase; the immunization then lasts 8 weeks.The serum is withdrawn after the immunization period and theimmunoglobulin may be isolated according to the method described byAxelsen N. H. (1983).

The present invention also relates to the identification and provisionof a new, isolated and purified aerobic bacterium producing xylanase.Generally, it belongs to the family Bacillaceae. Preferably, it belongsto the genus Bacillus. As a special preference, the said Bacillus isBacillus sp. strain 720/1 or a derivative or mutant of this strain.

Derivative of this strain is understood to mean any naturally modifiedbacterium. The derivatives of this strain may be obtained by knownmodification techniques such as culture on specific medium, ultravioletradiation or X-rays. Mutant of this strain is understood to mean anyartificially modified bacterium. The mutants of this strain may beobtained by known modification techniques such as exposure to mutagenicagents and genetic engineering techniques. These techniques are known toa person skilled in the art and are described, in particular, inSambrook et al., 1989, Chapter 15.

Bacillus sp. strain 720/1 was deposited at the collection named BelgianCoordinated Collections of Microorganisms (LMG culture collection, GhentUniversity, Microbiology Laboratory—K. L. Ledeganckstraat 35, B-9000Ghent, Belgium) in accordance with the Budapest Treaty under the numberLMG P-14798 on 9 Jun. 1994. The invention relates to an isolated andpurified culture of Bacillus sp. strain 720/1 and to a derived ormutated culture of the latter.

The strain of the present invention was identified by its biochemicalfeatures: aerobic Gram-positive bacterium which takes the form of a rod;it forms an endospore. It is oligosporogenous.

The invention also relates to the isolation and provision of a DNAmolecule comprising the nucleotide sequence (SEQ ID NO:1) which codesfor the mature xylanase of Bacillus sp. 720/1 (LMG P-14798) or amodified sequence derived from this sequence. Preferably, this DNAmolecule comprises the entire Bacillus sp. 720/1 xylanase gene. Entirexylanase gene (SEQ ID NO:10) is understood to mean at least thetranscription promoter(s), the signal sequence(s), the nucleotidesequence coding for the mature xylanase and the transcriptionterminator(s).

Modified sequence derived from the DNA molecule is understood to meanany DNA molecule obtained by modification of one or more nucleotides ofthe gene which codes for the xylanase of the invention. The techniquesof obtaining such sequences are known to a person skilled in the art,and are described, in particular, in Molecular Cloning—a laboratorymanual—Sambrook, Fritsch, Maniatis—second edition, 1989, Chapter 15.Usually, the modified sequence derived from the DNA molecule comprisesat least 70% homology with the nucleotide sequences [sic] (SEQ ID NO:1)of the gene which codes for the xylanase of the invention, that is tosay at least 70% of identical nucleotides having the same position inthe sequence. Preferably, the modified sequence derived from the DNAmolecule comprises at least 80% homology with the nucleotide sequence(SEQ ID NO:1) of the gene which codes for the xylanase of the invention.As a special preference, the modified sequence derived from the DNAmolecule comprises at least 90% homology with the nucleotide sequences[sic] (SEQ ID NO:1) of the gene which codes for the xylanase of theinvention.

The complete nucleotide sequence coding for the mature xylanase,together with its translation into amino acids (SEQ ID NO:2), is givenin FIG. 1 (FIGS. 1 a and 1 b).

Usually, the DNA molecule according to the invention comprises at leastthe nucleotide sequence (SEQ ID NO:4) which codes for the xylanaseprecursor or a modified sequence derived from this sequence. Thisnucleotide sequence (SEQ ID NO:4) comprises the nucleotide sequence (SEQID NO:1) coding for the mature xylanase of Bacillus sp. 720/1 (LMGP-14798) and its signal sequence (presequence) (SEQ ID NO:7).Preferably, this DNA molecule comprises the entire Bacillus sp. 720/1xylanase gene and, as a special preference, the nucleotide sequence (SEQID NO:10). The nucleotide sequence (SEQ ID NO:10) consists, in theamino-carboxy direction and from left to right, of the nucleotidesequence (SEQ ID NO:12) which comprises the xylanase promoter, thenucleotide sequence of the presequence (SEQ ID NO:7), the nucleotidesequence of the mature xylanase (SEQ ID NO:1) and the nucleotidesequence (SEQ ID NO:13) which comprises the xylanase terminator. FIG. 2(FIG. 2 a and FIG. 2 b) shows the nucleotide sequence of the gene codingfor the xylanase, together with its translation into amino acids (SEQ IDNO:11).

In a variant, the invention also relates to a DNA molecule whichcomprises the promoter derived from the gene which codes for Bacilluspumilus PRL B12 xylanase, a presequence and the nucleotide sequence (SEQID NO:1) which codes for Bacillus sp. 720/1 xylanase or a modifiedsequence derived from this sequence. In another variant, the inventionalso relates to a DNA molecule which comprises a promoter, thepresequence which codes for the signal peptide of Bacillus pumilus PRLB12 xylanase and the nucleotide sequence (SEQ ID NO:1) which codes forBacillus sp. 720/1 xylanase or a modified sequence derived from thissequence. Preferably, the invention relates to a DNA molecule whichcomprises the promoter (SEQ ID NO:26) derived from the gene which codesfor Bacillus pumilus PRL B12 xylanase, the presequence (SEQ ID NO:27)which codes for the signal peptide of Bacillus pumilus PRL B12 xylanaseand the nucleotide sequence (SEQ ID NO:1) which codes for Bacillus sp.720/1 xylanase or a modified sequence derived from this sequence.

The invention also relates to the promoter (SEQ ID NO:26) derived fromthe gene which codes for Bacillus pumilus PRL B12 xylanase. The sequenceof the promoter is illustrated in FIG. 11.

The invention also relates to the presequence (SEQ ID NO:27) which codesfor the signal peptide of Bacillus pumilus PRL B12 xylanase. Thecorresponding sequence of 27 amino acids has been identified (SEQ IDNO:29). This nucleotide sequence, together with its translation intoamino acids (SEQ ID NO:28), is illustrated in FIG. 12.

The method for obtaining and preparing the promoter derived from thegene which codes for Bacillus pumilus PRL B12 xylanase and of thepresequence which codes for the signal peptide of Bacillus pumilus PRLB12 xylanase is described in Example 17 and in FIG. 1 of European PatentApplication 0,634,490, which is incorporated by reference in thisapplication.

Bacillus pumilus strain PRL B12 was deposited at the ATCC collection(American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.,20852, USA) in accordance with the Budapest Treaty under the number ATCC55443 on 24 Jun. 1993.

The invention also relates to a mutated DNA molecule, and to the mutatedxylanase derived therefrom (for which the mutated DNA molecule codes),obtained by modification of the nucleotide sequence of the gene whichcodes for the xylanase defined above. The techniques of obtaining suchmutated xylanases are known to a person skilled in the art and aredescribed, in particular, in Molecular Cloning—a laboratorymanual—Sambrook, Fritsch, Maniatis—second edition, 1989, Chapter 15.

The present invention also relates to an expression vector orchromosomal integration vector containing a DNA molecule as definedabove. Generally, the expression vector or the chromosomal integrationvector contains the DNA molecule which comprises the nucleotide sequence(SEQ ID NO:1) which codes for Bacillus sp. 720/1 xylanase or a modifiedsequence derived from this sequence. Usually, the expression vector orthe chromosomal integration vector contains a DNA molecule whichcomprises the gene which codes for the xylanase or a modified sequencederived from this sequence. Preferably, the expression vector or thechromosomal integration vector contains the DNA molecule which comprisesthe nucleotide sequence (SEQ ID NO:10) which codes for Bacillus sp.720/1 xylanase or a modified sequence derived from this sequence. As aspecial preference, this vector is the expression vector pUBRD-720X11.Good results have also been obtained with the expression vectorpUBR-720X11.

A variant of the invention relates to an expression vector or achromosomal integration vector which contains a DNA molecule comprisingthe gene which codes for the mature portion of the xylanase or amodified sequence derived from this molecule. Generally, the expressionvector or the chromosomal integration vector contains the DNA moleculewhich comprises the nucleotide sequence (SEQ ID NO:1) which codes forBacillus sp. 720/1 xylanase or a modified sequence derived from thissequence. Usually, the expression vector or the chromosomal integrationvector contains a DNA molecule which comprises the promoter derived fromthe gene (SEQ ID NO:26) which codes for Bacillus pumilus PRL B12xylanase, a presequence and the nucleotide sequence (SEQ ID NO:1) whichcodes for Bacillus sp. 720/1 xylanase or a modified sequence derivedfrom this sequence. In a usual variant, the expression vector contains aDNA molecule which comprises a promoter, the presequence (SEQ ID NO:27)which codes for the signal peptide of Bacillus pumilus PRL B12 xylanaseand the nucleotide sequence (SEQ ID NO:1) which codes for Bacillus sp.720/1 xylanase or a modified sequence derived from this sequence.Preferably, the expression vector or the chromosome integration vectorcontains a DNA molecule which comprises the promoter derived from thegene (SEQ ID NO:26) which codes for Bacillus pumilus PRL B12 xylanase,the presequence (SEQ ID NO:27) which codes for the signal peptide ofBacillus pumilus PRL B12 xylanase and the nucleotide sequence (SEQ IDNO:1) which codes for Bacillus sp. 720/1 xylanase or a modified sequencederived from this sequence. As a special preference, this vector is theexpression vector pBPXD-PRE-720X. Good results have also been obtainedwith the expression vector pC-BPX-PRE-720X.

The invention also relates to an expression system which can be used forthe production of a polypeptide.

This expression system comprises:

-   -   the sequence of the promoter (SEQ ID NO:26) derived from the        gene which codes for Bacillus pumilus PRL B12 xylanase,    -   a sequence coding for a signal peptide, and    -   the sequence of the polypeptide of interest.

Generally, the expression system comprises the sequence of a terminator.

In a variant, this expression system comprises:

-   -   the sequence of a promoter,    -   the presequence (SEQ ID NO:27) which codes for the signal        peptide of Bacillus pumilus PRL B12 xylanase, and    -   the sequence of the polypeptide of interest.

Generally, the expression system comprises the sequence of a terminator.

Usually, this expression system comprises:

-   -   the sequence of the promoter (SEQ ID NO:26) derived from the        gene which codes for Bacillus pumilus PRL B12 xylanase,    -   the presequence (SEQ ID NO:27) which codes for the signal        peptide of Bacillus pumilus PRL B12 xylanase,    -   the sequence of the polypeptide of interest, and    -   the sequence of a terminator.

Preferably, the polypeptide of interest is an enzyme such as ahydrolase. As a special preference, the polypeptide of interest is aprotease, a lipase, a xylanase, a cellulase, an amylase or apullulanase. Good results have been obtained with the xylanase naturallyproduced by Bacillus sp. strain 720/1, that is to say when, in theexpression system, the sequence of the polypeptide corresponds to thenucleotide sequence (SEQ ID NO:1) which codes for Bacillus sp. 720/1xylanase.

The present invention also relates to recombinant strains into which thegene coding for a xylanase is introduced by genetic engineeringtechniques. The gene may be introduced by means of a replicative vector,or integrated in the host's chromosome in one or more copies by means ofan integrative vector; the nucleotide sequence coding for a xylanase maybe introduced by transformation, either in integrated form in thechromosomal DNA, or in self-replicating form (plasmid).

The invention also relates to strains of microorganisms which aredifferent from the initial producer organism, into which strains thenucleotide sequence coding for a xylanase is introduced bytransformation, either in integrated form in the chromosomal DNA, or inself-replicating form (plasmid); the gene coding for a xylanase may beintroduced by means of a replicative vector or integrated in the host'schromosome in one or more copies by an integrative vector.

The invention relates to a transformed strain comprising the DNAmolecule which contains the structural gene which codes for the maturexylanase of Bacillus sp. 720/1. Generally, the transformed strain is astrain of bacterium. Usually, the transformed strain is chosen fromEscherichia, Pseudomonas or Bacillus strains. Preferably, thetransformed strain is a Bacillus strain. As a special preference, thetransformed Bacillus strain is a Bacillus licheniformis strain, aBacillus pumilus strain, a Bacillus alcalophilus strain or a Bacillussp. strain 720/1. Good results have been obtained with a Bacilluslicheniformis strain and with a Bacillus pumilus strain.

The invention relates to the transformed Bacillus strain comprising theexpression vector or the chromosomal integration vector which comprisesthis DNA molecule. Preferably, the transformed Bacillus strain is aBacillus licheniformis strain. Preferably also, the transformed Bacillusstrain is a Bacillus sp. strain 720/1.

The present invention also relates to the xylanase produced by atransformed strain as defined above.

The present invention also relates to a method for the production of axylanase, comprising the culturing of an aerobic bacterium capable ofproducing the xylanase in a suitable nutrient medium containing carbonand nitrogen sources and inorganic salts under aerobic conditions, andthe harvesting of the xylanase thereby obtained. This culture medium canbe solid or liquid. Preferably, the culture medium is liquid.Preferably, the aerobic bacterium is a Bacillus strain or a derivativeof this strain capable of producing the xylanase.

The present invention also relates to a method for the production of axylanase, comprising the culturing of Bacillus sp. strain 720/1 or aderivative of this strain capable of producing the xylanase in asuitable nutrient medium containing carbon and nitrogen sources andinorganic salts under aerobic conditions, and the harvesting of thexylanase thereby obtained.

The invention also relates to a method for the preparation of a xylanasefrom a recombinant organism, the method comprising the isolation of aDNA fragment coding for the xylanase, the insertion of this DNA fragmentinto a suitable vector, the introduction of this vector into a suitablehost or the introduction of this DNA fragment into the chromosome of asuitable host, the culturing of this host, the expression of thexylanase and the harvesting of the xylanase. The suitable host isgenerally chosen from the group consisting of Escherichia coli, Bacillusor Aspergillus microorganisms. Usually, the host is chosen from Bacillusspecies. Preferably, the host is chosen from microorganisms of the genusBacillus (aerobic). As a special preference, the host is chosen from themicroorganisms Bacillus subtilis, Bacillus licheniformis, Bacillusalcalophilus, Bacillus pumilus, Bacillus lentus, Bacillusamyloliquefaciens or Bacillus sp. 720/1. Good results have been obtainedwhen the host for the expression of the xylanase according to thepresent invention is a recombinant strain derived from Bacilluslicheniformis, and preferably Bacillus licheniformis strain SE2 delap1and Bacillus licheniformis strain SE2 delap6. Bacillus licheniformisstrain SE2 delap1 and Bacillus licheniformis strain SE2 delap6 aredescribed in European Patent Application 0,634,490, which isincorporated by reference in this application.

The invention also relates to a xylanase produced heterologously by amicroorganism of the genus Bacillus. Usually, the microorganism of thegenus Bacillus contains a gene coding for an alkaline protease when itis in the wild-type state. Preferably, this microorganism is a Bacilluslicheniformis strain comprising the DNA molecule which comprises thenucleotide sequence which codes for Bacillus sp. 720/1 xylanase. As aspecial preference, the gene coding for the alkaline protease has beenremoved by deletion from this Bacillus strain. This strain is preferablyBacillus licheniformis strain SE2 delap1 or Bacillus licheniformisstrain SE2 delap6.

Produced heterologously is understood to mean a production which is notperformed by the natural microorganism, that is to say the microorganismwhich, in the wild-type state, contains the gene which codes for thexylanase.

The conditions of culture of these bacteria, such as components of thenutrient medium, culture parameters, temperature, pH, aeration andagitation, are well known to a person skilled in the art. Examples ofsuch techniques are described, in particular, in Ullmann's Encyclopediaof Industrial Chemistry, 1987, 5th Edition, Vol. A9, pages 363-390.

The techniques of harvesting of xylanase are well known to a personskilled in the art, and are chosen according to the uses envisaged forthe xylanase. Usually, centrifugation, filtration, ultrafiltration,evaporation, microfiltration, crystallization or a combination of one orother of these techniques is used, such as a centrifugation followed byan ultrafiltration. Examples of such techniques are described, inparticular, by R. Scriban, Biotechnology, (Technique et DocumentationLavoisier), 1982, pp. 267-276 and in Ullmann's Encyclopedia ofIndustrial Chemistry, 1987, 5th Edition, Vol. A9, pages 363-390.

The xylanase can then be purified, if necessary and according to theuses envisaged. Enzyme purification techniques are well known to aperson skilled in the art, such as precipitation using a salt such asammonium sulphate, or using a solvent such as acetone or an alcohol.Examples of such techniques are described, in particular, by R. Scriban,Biotechnology, (Technique et Documentation Lavoisier), 1982, pp.267-276.

The xylanase may also be dried by atomization or lyophilization.Examples of such techniques are described, in particular, by R. Scriban,Biotechnology, (Technique et Documentation Lavoisier), 1982, pp. 267-276and in Ullmann's Encyclopedia of Industrial Chemistry, 1987, 5thEdition, Vol. A9, pages 363-390.

The present invention also relates to enzyme compositions comprising thexylanase according to the invention and at least one additive. Theseadditives are known to a person skilled in the art and are chosenaccording to the use envisaged for the composition. They must becompatible with the xylanase and must have little or no effect on theenzyme activity of the xylanase. Usually, these additives are enzymestabilizers, preservatives and formulation agents.

The compositions comprising the xylanase of the present invention may beused in solid or liquid form.

The xylanase is formulated according to the anticipated uses.Stabilizers or preservatives may also be added to the enzymecompositions comprising the xylanase according to the invention. Forexample, it is possible to stabilize the xylanase by adding propyleneglycol, ethylene glycol, glycerol, starch, xylan, a sugar such asglucose and sorbitol, a salt such as sodium chloride, calcium chloride,potassium sorbate and sodium benzoate or a mixture of two or more ofthese products. Good results have been obtained with propylene glycol.Good results have been obtained with sorbitol.

The xylanase according to the invention has numerous outlets in variousindustries such as, for example, the food industries, the pharmaceuticalindustries or the chemical industries.

The xylanase may be used, in particular, in bakery. An example of use ofa xylanase in bakery is described, in particular, in InternationalPatent Application WO 94/04664.

The xylanase can be used, in particular, for the treatment of paperpulp. An example of the use of a xylanase for the treatment of paperpulp is described, in particular, in European Patent Application0,634,490. The xylanase of the present invention is effective, inparticular, on the pulp originating from eucalyptus wood, as illustratedin Example 13 of the present patent application.

The xylanase can be used, in particular, in animal feeds. An example ofa use of a xylanase in animal feeds is described, in particular, inEuropean Patent Application 0,507,723.

FIG. 1 (FIG. 1 a and FIG. 1 b) shows the nucleotide sequence (SEQ IDNO:2) coding for the mature xylanase, together with its translation intoamino acids.

FIG. 2 (FIG. 2 a and FIG. 2 b) shows the nucleotide sequence (SEQ IDNO:11) of the gene coding for xylanase, together with its translationinto amino acids.

FIG. 3 shows the restriction map of plasmid pUBR2002.

FIG. 4 shows the restriction map of plasmid pUBR-720×1.

FIG. 5 shows the restriction map of plasmid pUBR-720X11.

FIG. 6 shows the restriction map of plasmid pUBRD-720X11.

FIG. 7 shows the restriction map of plasmid pUBC2001.

FIG. 8 shows the restriction map of plasmid pC-BPX-PRE-2003

FIG. 9 shows the restriction map of plasmid pC-BPX-PRE-720X.

FIG. 10 shows the restriction map of plasmid pBPXD-PRE-720X.

FIG. 11 shows the promoter (SEQ ID NO:26) derived from the gene whichcodes for Bacillus pumilus PRL B12 xylanase.

FIG. 12 shows the presequence (SEQ ID NO:28) which codes for the signalpeptide of Bacillus pumilus PRL B12 xylanase.

The meaning of the abbreviations and symbols used in these figures iscollated in the following table.

Symbol Abbreviation Meaning OriEC origin of replication in E. coli REPprotein needed for replication in Bacillus Ori+ origin of replication ofthe + strand in Bacillus Ori− origin of replication of the − strand inBacillus AmpR gene conferring resistance to ampicillin KmR geneconferring resistance to kanamycin BlmR gene conferring resistance tobleomycin 5′720XYL 5′ sequence located upstream of the sequence codingfor Bacillus sp. 720/1 xylanase 3′720XYL 3′ sequence located downstreamof the sequence coding for Bacillus sp. 720/1 xylanase 720XYL sequencecoding for the Bacillus sp. 720/1 xylanase precursor 5′BPUXYL-PREpromoter and ribosome binding site of Bacillus pumilus PRL B12 xylanase,followed by the presequence of Bacillus pumilus PRL B12 xylanase720XYL-MAT sequence coding for the mature portion of Bacillus sp. 720/1xylanase

The present invention is illustrated by the examples which follow.

EXAMPLE 1 Isolation and Characterization of Bacillus sp. Strain 720/1

Bacillus sp. strain 720/1 was isolated from a sample of soil, obtainedin Argentina, on a nutrient agar medium, and selected for its capacityto degrade a coloured xylan derivative known by the name of AZCL-xylanand sold by the company Megazyme.

This strain was cultured at 37° C. in LBS/C growth medium whosecomposition is as follows: wheat bran 10 g/l, Tryptone (Difco) 10 g/l,yeast extract 5 g/l, NaCl 10 g/l, Na₂CO₃ 5.3 g/l, NaHCO₃ 4.2 g/l.

The sodium carbonate and bicarbonate are sterilized separately and thenadded aseptically to the other components of the sterile medium. Theagar medium contains, in addition, 20 g/l of agar. The strain of thepresent invention was identified by its biochemical features: aerobicGram positive bacterium which takes the form of a rod; it forms anendospore. Hence it belongs to the genus Bacillus.

The vegetative cells of this strain in culture on LBS/C agar medium at37° C. have a bacillus shape 0.8×3.0-5 μm in size. The mobility of thevegetative cells is positive.

After growth for 13 days at 37° C. on TSA agar medium, microscopicobservation reveals the presence of sporangia. TSA agar medium contains15 g/l of Tryptone (Difco), 5 g/l of soya bean peptone, 5 g/l of NaCland 15 g/l of agar.

The strain is oligosporogenous.

The test for catalase is positive in the presence of 10% (v/v) ofhydrogen peroxide. The test for oxydase is positive in the presence of1% (w/v) of tetramethyl-1,4-phenylenediammonium dichloride.

This strain is aerobic, that is to say grows under aerobic conditions.It does not grow under anaerobic conditions, that is to say under anatmosphere of 84% (v/v) N₂, 8% (v/v) CO₂, 8% (v/v) H₂ at 37° C. Theabbreviation % (v/v) represents a percentage expressed in terms ofvolume per volume.

This strain is not thermophilic. It displays normal growth afterincubation in LBS/C agar medium at 20° C., 30° C., 37° C. and 45° C.; incontrast, it does not grow at 50° C. and 55° C., or at 10° C.

It displays normal growth after incubation in LBS/C agar medium in thepresence of NaCl at concentrations of 2.0% (w/v) and 3.5% (w/v), anddisplays slight growth in the presence of 5.0% (w/v) and 7.0% (w/v)NaCl. The abbreviation % (w/v) represents a percentage expressed interms of weight per volume.

Bacillus sp. strain 720/1 does not acifify glucose.

Bacillus sp. strain 720/1 has been identified by means of the API 50 CHBstrip and the API 20 E strip following the instructions for use of thesupplier (API System, France). Bacillus sp. strain 720/1 utilisesglycerol, N-acetylglucosamine, arbutin, citrate, galactose, amygdalinand melibiose, and hydrolyses gelatin. These features differentiateBacillus sp. strain 720/1 clearly from a Bacillus pumilus strain. Ineffect, a Bacillus pumilus strain does not display any of thesefeatures.

Bacillus sp. strain 720/1 was also identified by means of the Biologsystem (USA). The data bank analysing the results of this system gives ascore of 0.564 for Bacillus coagulans, 0.097 for Bacillus subtilis,0.057 for Bacillus licheniformis and 0.00 for Bacillus pumilus. Thesefeatures differentiate Bacillus sp. strain 720/1 clearly from a Bacilluscoagulans strain, a Bacillus subtilis strain, from a Bacilluslicheniformis strain and from a Bacillus pumilus strain.

Hence the isolated bacterium belongs to the genus Bacillus; no knownspecies could be determined.

Bacillus sp. strain 720/1 was deposited at the collection named BelgianCoordinated Collections of Microorganisms (LMG culture collection) underthe number LMG P-14798.

EXAMPLE 2 Production of Xylanase by Bacillus sp. 720/1

Bacillus sp. strain 720/1 is cultured on Petri dishes containing anLBS/C agar medium at 37° C. for 48 hours (culture A).

Then, from the culture A, culturing is carried out in an LB/C liquidmedium whose composition is identical to that of LBS/C medium butwithout wheat bran, at 37° C. for 24 hours with orbital shaking at therate of 250 rpm (culture B) with an amplitude of approximately 2.54 cm.

500 ml of the culture B are then transferred to a 20-1 fermentercontaining 14 l of LBS/C medium. The pH is allowed to find its naturalvalue, and the speed of agitation and the flow rate of air blown intothe fermenter are such that the partial pressure of oxygen dissolved inthe culture medium is not below 30% of the saturation value.

After 72 hours of culture at 37° C., the xylanase and the cellularbiomass are separated by centrifugation (Beckman J21, JA10 rotor) at8,000 rpm for 30 minutes. The xylanase produced by Bacillus sp. strain720/1 is extracellular. From the centrifugation supernatant, theresidual insoluble matter is then separated from the xylanase bymicrofiltration (KROS FLOII cartridge, porosity 0.2μ, company Microgon).

The microfiltration retentate is washed with 1 l of demineralized water.This washing is performed three times.

The permeate of this microfiltration is then concentrated approximately20-fold by ultrafiltration through a Pall MICROZA SIP 1013 polysulphonecartridge having a cut-off threshold of 6 kD (company Pall).

The enzyme activity is measured on the ultrafiltration retentate(product R) and permeate (product P) obtained.

One xylanase enzyme unit (IU) is defined as the amount of enzyme which,at pH 8.0, at a temperature of 50° C. and in the presence of xylan,catalyses the liberation of glucose equivalents at the rate of 1 μmol ofglucose per minute (μM [sic]/minute).

The measurement of xylanase enzyme activity is carried out according tothe protocol described by Bailey, Biely and Poutanen, J. Biotechnology,1992, 23, pages 257-270; except that the citrate-phosphate buffermentioned by Bailey et al. was replaced by 50 mMtris(hydroxymethyl)aminoethane-HCl buffer (pH 8.0).

Sufficient polyethylene glycol (Merck polyethylene glycol reference807490) is added to the ultrafiltration retentate (product R) to obtaina concentration of 40% (w/w). After solubilization of the polyethyleneglycol, the solution obtained is incubated for 30 minutes at 25° C.

The solution containing the polyethylene glycol and the xylanase is thencentrifuged for 10 minutes at 8,000 rpm (Beckman J21 centrifuge, JA10rotor). The supernatant is removed by centrifugation. Sufficient NaClsolution (0.9% v/v) is added to the centrifugation pellet to recover theinitial volume of the retentate used (product R).

Sufficient acetone is then added to this suspension containing thexylanase and NaCl to achieve a concentration of 40% (v/v). This acetonesuspension is incubated for 45 minutes at 4° C.

After this incubation, this acetone suspension is centrifuged for 10minutes at 8,000 rpm (Beckman J21, JA10 rotor).

The centrifugation supernatant is retained. To this centrifugationsupernatant, acetone is added to a concentration of 80% (v/v). Thisacetone suspension is incubated for 45 minutes at 4° C.

After this incubation, this acetone suspension is centrifuged for 10minutes at 8,000 rpm (Beckman J21, JA10 rotor).

The centrifugation pellet is retained. It is suspended in a sufficientvolume of 0.9% (v/v) NaCl solution to be solubilized (product N).

EXAMPLE 3 Purification of the Xylanase

A fraction of the ultrafiltration retentate (product N) obtained inExample 2 is conditioned by passage through a gel permeationchromatography column (Bio-Rad Econopac 10DG column) equilibrated with20 mM Bio-Tris(bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane)buffer, pH 6.2 (buffer A). A solution designated product X is therebyobtained.

1 ml of the product X solution is then applied to an S Sepharose HP16/10 (Pharmacia) cation exchange column previously equilibrated withthe buffer A. The flow rate is 2.5 ml per minute, with an isocraticelution for 10 minutes, followed by an NaCl concentration gradient (from10 to 50 minutes; the NaCl content rises from 0 to 0.7 M). A single peakis detected during the gradient, corresponding to the elution of thexylanase.

The fractions containing the xylanase activity (solution A) arecollected.

It is verified that these fractions contain xylanase by applying 10 μlof each fraction to an agar medium comprising xylan (medium containing0.5 g/l of AZCL-xylan, 50 mM Tris buffer (pH 8.0) and 15 g/l of agar). Ahalo forms around the fractions which contain xylanase.

EXAMPLE 4 Amino Acid Sequence

The amino acid sequence of the xylanase of the present invention isdetermined indirectly from the nucleotide sequence (SEQ ID NO:10) of thegene which codes for this xylanase, which is obtained as described inExample 14. This is carried out using the IntelliGenetics Suite Softwarefor Molecular Biology (Release #5.4) computer program ofIntelliGenetics, Inc. USA. FIG. 2 (FIG. 2 a and FIG. 2 b) shows thenucleotide sequence (SEQ ID NO:10) of the gene coding for the xylanase,together with its translation into amino acids (SEQ ID NO:11).

The xylanase is synthesized in the form of a precursor. The xylanaseprecursor contains 248 amino acids (SEQ ID NO:6). The nucleotidesequence (SEQ ID NO:4) coding for the xylanase precursor, as well as itstranslation into amino acids (SEQ ID NO:5), are identified.

The presequence of the xylanase synthesized in the form of a precursoris identified. It is a sequence of 27 amino acids (SEQ ID NO:9). Thecorresponding nucleotide sequence (SEQ ID NO:7) is identified.

The amino acid sequence of the mature xylanase is then identified. Themature xylanase contains 221 amino acids (SEQ ID NO:3).

FIG. 1 (FIG. 1 a and FIG. 1 b) shows the nucleotide sequence (SEQ IDNO:1) coding for the mature xylanase, together with its translation intoamino acids (SEQ ID NO:2).

EXAMPLE 5 Amino Acid Distribution

The amino acid distribution of the mature xylanase, determined from theamino acid sequence (SEQ ID NO:3) of the xylanase (Example 4), issummarized in Table 1.

TABLE 1 % (in molecular Symbol Amino acid Number weight) N asparagine 2511.6 Y tyrosine 13 8.6 T threonine 18 7.4 S serine 19 6.7 I isoleucine14 6.4 V valine 14 5.6 G glycine 24 5.5 W tryptophan 7 5.3 K lysine 105.2 R arginine 8 5.1 Q glutamine 9 4.7 D aspartic acid 10 4.7 L leucine10 4.6 E glutamic acid 8 4.2 F phenylalanine 7 4.2 P proline 7 2.8 Mmethionine 5 2.7 A alanine 8 2.3 H histidine 4 2.2 C cysteine 1 0.4 Baspartic acid/asparagine 0 0.0 X unknown 0 0.0 Z glutamine glutamic acid0 0.0

EXAMPLE 6 Estimation of the Molecular Weight

The molecular weight of the xylanase is estimated by calculation fromthe amino acid sequence of the mature form of the xylanase, as describedin Example 4.

A molecular weight of 24698.61 daltons is deduced by calculation.

EXAMPLE 7 Molecular Weight Determination

Concentration on a Centricon 10 kD device (Amicon) is performed on thesolution A containing the xylanase, as obtained in Example 3.

100 μl of the concentrated solution are applied to a Superdex 75 HR10/30 (Pharmacia) gel permeation chromatography column. The column waspreviously calibrated by means of the (Pharmacia) Gel Filtration LMWcalibration kit, code 17-0442-01, molecular weight markers. Elution tookplace at 0.25 ml/minute by means of 25 mM CAPSO(3-(cyclohexylamino)-2-hydroxy-1-propanesulphonic acid) buffer pH 9.2,with the addition of 0.2 M NaCl.

The chromatogram obtained shows a single peak corresponding to thexylanase activity. An apparent molecular weight of the protein ofapproximately 13.5 kD is deduced from this.

Polyacrylamide gel electrophoresis under denaturing conditions(SDS-PAGE) is also performed on the fraction originating from thissingle peak. The gel system used is the PhastSystem system of PharmaciaLKB Biotechnology, with gels containing a polyacrylamide gradient from10-15% (v/v). Electrophoresis conditions are those prescribed by thesupplier. Pharmacia LMW (Low Molecular Weight) molecular weight markers,reference 17-0446-01, are used as control. The markers employed arephosphorylase b (94 kD), albumin (67 kD), ovalbumin (43 kD), carbonicanhydrase (30 kD), trypsin inhibitor (20.1 kD) and alpha-lactalbumin(14.4 kD).

Staining with Coomassie blue reveals a polypeptide of molecular weightapproximately 25.7 kD.

EXAMPLE 8 Estimation of the Isoelectric Point

The isoelectric point of the xylanase is estimated from the amino acidsequence of the mature form of the xylanase, as described in Example 4.

The estimated isoelectric point represents the net charge of the proteinin denatured form.

An isoelectric point of 7.46 is deduced for the xylanase in denaturedform.

EXAMPLE 9 Isoelectric Point Determination

A fraction of the solution A, as obtained in Example 3, is applied to aMono P 5/20 chromatofocusing column (Pharmacia), following thesupplier's recommendations, previously equilibrated with a 25 mMdiethanolamine buffer, pH 9.9.

The column is eluted by means of the Polybuffer 96 ampholyte solution(Pharmacia) diluted 10-fold in demineralized water.

The pH of the fraction containing the xylanase activity is 9.5.

It is verified that this fraction contains xylanase by applying 10 μl ofthe fraction to an agar medium comprising xylan (medium containing 0.5g/l of AZCL-xylan, 50 mM Tris buffer (pH 8.0) and 15 g/l of agar). Ahalo, which takes the form of a zone of hydrolysis of AZCL-xylan, formsaround the fraction which contains the xylanase.

Isoelectric focusing is carried out on a fraction of the solution A asobtained in Example 3.

To do this, Pharmacia DryIEF gel is rehydrated with a mixture consistingof 2 ml of demineralized water, 150 μl of Biolyte 8-10 product (BioRad)and 75 μl of Pharmalyte 8-10.5 product (Pharmacia). Approximately 200nanogrammes of proteins (fraction of the solution A) are applied to thegel. The protocol described by the supplier is followed.

It is deduced from this that the xylanase has an isoelectric pointslightly above 9.6, the isoelectric point of the marker having thehighest isoelectric point used.

The experimentally observed isoelectric point represents the surfacecharge of the protein in its native form.

EXAMPLE 10 Activity Profile as a Function of pH for the XylanaseProduced by the Natural Strain (Bacillus sp. 720/1)

The enzyme activity of the xylanase is measured according to the methoddescribed in Example 2 in the presence of xylan (Roth, reference 7500,birchwood xylan) at a temperature of 50° C. and at different pH values(from 5.6 to 10.35) in different buffers chosen to obtain the desiredpH. The solution comprising the xylanase as obtained in Example 2(product N) is employed.

The results are collated in Table 2. It may be noted that the margin oferror is estimated at approximately 25% in this type of measurement.

In the course of this assay, maximal enzyme activity was measured forthe sample placed at a pH of approximately 6.2 and at a temperature ofapproximately 50° C. for 15 minutes. By definition, a relative enzymeactivity of 100% was hence assigned to this sample.

This example shows that the xylanase according to the invention developsconsiderable enzyme activity over a pH range between approximately 5.6and approximately 10.

TABLE 2 Buffer used Relative pH (50 mM) activity % 5.6 Tris-maleate 856.2 Tris-maleate 100 6.8 Tris-maleate 96 7.5 Tris- 88 maleate/Tris* 8.7Tris/Capso* 92 9.5 Capso/Caps* 76 10 Caps 50 10.35 Caps 19 Tris =tris(hydroxymethyl)aminomethane Tris-maleate = buffer composed oftris(hydroxymethyl)aminomethane (50 mM) and maleic acid (50 mM), inwhich the ratio between the components is chosen in accordance with thedesired pH, the pH being adjusted by means of NaOH (1 M) Capso =3-(cyclohexylamino)-2-hydroxy-1-propanesulphonic acid Caps =3-(cyclohexylamino)-1-propanesulphonic acid The symbol * means that thevalue obtained is the mean of the measurements performed at the same pHbut obtained with the two buffers.

This example shows that the xylanase according to the invention developshigh enzyme activity over a very wide pH range.

EXAMPLE 11 Effect of pH on the Activity of the Xylanase Produced byBacillus sp. Strain 720/1 as an Aid to the Bleaching of Coniferous WoodPulp

Three aqueous suspensions of a pinewood pulp (obtained from the companySCA) are prepared, having a consistency of 2.5% (as weight of drymatter) and having an initial Kappa number of 17.

The pH of these suspensions is adjusted to pH 5

1st Stage: Enzyme Stage (Stage X)

The solution designated product N, as obtained in Example 2, is dilutedwith demineralized water to obtain an enzyme solution having an enzymeactivity of 25 IU/ml (as described in Example 2).

This enzyme solution containing the xylanase is added to one suspensionof pinewood pulp such that the pulp suspension is treated by means of 5IU/g of dry pulp.

To the other two suspensions of pinewood pulp, demineralized water isadded in place of the enzyme solution in identical proportions.

The three suspensions are then incubated for 2 hours at 50° C. withoutstirring.

2nd Stage: Chlorine Stage (Stage C)

Each pulp suspension thereby obtained is then subjected to a bleachingtreatment which consists of a chlorination with chlorine water. Thistreatment takes place on a pulp having a consistency of 3% as weight ofdry matter.

To this end, an amount of chlorine of 2.89 (=0.17×17) % (weight/weightof dry pulp) is added to the enzyme-treated pulp suspension and to onenon-enzyme-treated pulp suspension. An amount of chlorine of 3.40(0.20×17) % (weight/weight) is added to the other non-enzyme-treatedpulp suspension.

The 3 suspensions are incubated for 1 hour at room temperature. The pulpis then washed with 40 volumes of demineralized water.

3rd Stage: Sodium Hydroxide Stage (Stage E)

An alkaline extraction is then performed, which consists in adding 2%(weight/weight of dry pulp) of NaOH to the three suspensions obtainedabove and which have a consistency of 5% as weight of dry matter.

The three suspensions are incubated for 1 hour 30 minutes at atemperature of 60° C. The pulp is then washed with 40 volumes ofdemineralized water and recovered in the form of a sheet having awhiteness of approximately 450 ISO (+/−3° ISO).

The Kappa number of the three sheets obtained is measured.

The Kappa number relates to the measurement of the amount of ligninpresent in the pulp. The Kappa number is a number which represents thevolume (in milliliters) of 0.1 N potassium permanganate (KMnO₄) solutionconsumed by one gram of dry pulp under the conditions specified andfollowing the procedures described in TAPPI (Technical Committee of theAssociation of the Pulp and Paper industry) standard #T236 cm-85 (1985).

The degree ISO relates to the measurement of brightness of the paperobtained from the pulp. This value is a factor of the reflectance of thepaper obtained from the pulp under the conditions specified andfollowing the procedures described in ISO (The InternationalOrganization for Standardization) standard #2469 published in standard#ISO 2470-1977 (F) supplementing standard #2470.

Four assays are carried out, which are identical except for the initialpH adjustment to pH 5. In effect, four assays are carried out with anaqueous suspension of pinewood pulp whose pH has been adjusted,respectively, to pH 6, pH 7, pH 8 and pH 9 instead of pH 5.

The results are collated in Table 3.

TABLE 3 Amount of enzymes 5 0 0 employed (IU/g) Amount of chlorine 2.892.89 3.40 employed (% by weight/weight of dry pulp) Initial pH Kappanumber 5 4.63 5.01 4.18 6 3.81 / 4.11 7 3.81 5.01 4.06 8 3.55 / 4.21 93.71 4.91 4.07 The symbol / means that the pulp suspension was nottested.

These results show that the xylanase according to the invention permitsan approximately 15 to 20% reduction in the amount of chlorine for apulp bleached to 450 ISO. Furthermore, these results are obtained bothat an alkaline pH and at an acid pH. These good results are alsoobtained at a pH of approximately 9.

This example shows that the xylanase according to the invention displaysactivity over a wide pH range. In effect, the xylanase according to theinvention is active over a pH range between approximately 5 andapproximately 10. It is especially active for pH values above or equalto approximately 6. It is especially active for pH values below or equalto approximately 9.

EXAMPLE 12 Effect of Temperature on the Activity of the XylanaseProduced by Bacillus sp. Strain 720/1 as an Aid to the Bleaching ofConiferous Wood Pulp

Example 11 is repeated with 5 suspensions of coniferous wood pulp at pH8. The enzyme treatment stage is carried out at a pH of 8 and atdifferent temperatures (55, 60 and 65° C.).

The results are collated in Table 4.

TABLE 4 Amount of enzymes 5 0 0 employed (IU/g) Amount of chlorine 2.892.89 3.40 employed (% by weight/weight of dry pulp) Temperature ° C.Kappa number 55 3.89 / / 60 3.64 4.77 4.09 65 3.49 / / The symbol /means that the pulp suspension was not tested.

It is observed that the pulp is bleached to approximately 45° ISO.

These results show that the Kappa number of the enzyme-treated pulpsamples remains well below the number of the non-enzyme-treated sample.

This example shows that the xylanase according to the invention isactive over a wide temperature range. It is active at a temperature ofapproximately 65° C.

This example also shows that the xylanase according to the invention isstable at a temperature of approximately 60° C.

EXAMPLE 13 Activity of the Xylanase Produced by Bacillus sp. Strain720/1 as an Aid to the Bleaching of Eucalyptus Pulp in the ECF Sequence

For this example, a eucalyptus pulp obtained from the company CEASA Mill(Spain) is employed. The pulp is treated according to an ECF (“ElementalChlorine Free”) sequence, that is to say the succession of stagesconstituting the sequence does not make use of elemental chlorine.

1st Stage: Oxygen Stage (Stage O)

The pulp is treated by a process employing oxygen as described in U.S.Pat. No. 4,462,864, such that a pulp having an initial Kappa number of12.3 and an initial degree ISO of 33.4 is obtained.

Two aqueous suspensions are prepared from this oxygen-treated pulphaving a consistency of 4% as weight of dry matter.

The pH of these suspensions is adjusted to pH 9 with HCl.

2nd Stage: Enzyme Stage (Stage X)

The solution designated product N, as obtained in Example 2, is dilutedwith demineralized water to obtain an enzyme solution having an enzymeactivity of 25 IU/ml.

This enzyme solution containing the xylanase is added to one suspensionof pinewood pulp such that the pulp suspension is treated by means of 10IU/g of dry pulp.

To the other two suspensions of pinewood pulp, demineralized water isadded in place of the enzyme solution.

The 3 suspensions are then incubated for 1 hour 30 minutes at 50° C.without stirring.

3rd Stage: Chlorine Dioxide Stage (Stage D)

Each pulp suspension thereby obtained is then subjected to a bleachingtreatment which consists of a chlorination with chlorine dioxide. Thistreatment takes place on a pulp having a consistency of 3% as weight ofdry matter.

To this end, an amount of chlorine dioxide of 0.6% (weight/weight of drypulp) is added to the enzyme-treated pulp suspension and to onenon-enzyme-treated pulp suspension. An amount of chlorine dioxide of 1%(weight/weight) is added to the other non-enzyme-treated pulpsuspension.

The 3 suspensions are incubated for 30 minutes at 50° C. The pulp isthen washed with 40 volumes of demineralized water.

4th Stage: Sodium Hydroxide/Hydrogen Peroxide Stage (Stage E/P)

An alkaline extraction is then performed, which consists in adding 1.8%(weight/weight of dry pulp) of NaOH and 0.5% (weight/weight of dry pulp)of hydrogen peroxide to the three suspensions obtained above, and whichhave a consistency of 12% as weight of dry matter.

The three suspensions are incubated for 1 hour 30 minutes at atemperature of 70° C. The pulp is then washed with 40 volumes ofdemineralized water.

5th Stage: Chlorine Dioxide Stage (Stage D)

Each pulp suspension thereby obtained is then subjected again to ableaching treatment which consists of a chlorination with chlorinedioxide. This treatment takes place on a pulp having a consistency of12%.

An amount of chlorine dioxide of 0.5% (weight/weight of dry pulp) isadded to these three suspensions.

The 3 suspensions are incubated for 2 hours at 75° C. The pulp is thenwashed with 40 volumes of demineralized water.

6th Stage: Sodium Hydroxide/Hydrogen Peroxide Stage (Stage E/P)

An alkaline extraction is then performed, which consists in adding 0.6%(weight/weight of dry pulp) of NaOH and 0.3% (weight/weight of dry pulp)hydrogen peroxide to the three suspensions obtained above, and whichhave a consistency of 12% as weight of dry matter.

The three suspensions are incubated for 1 hour 30 minutes at atemperature of 70° C. The pulp is then washed with 40 volumes ofdemineralized water.

7th Stage: Chlorine Dioxide Stage (Stage D)

Each pulp suspension thereby obtained is then subjected to a bleachingtreatment which consists of a chlorination with chlorine dioxide. Thistreatment takes place on a pulp having a consistency of 12%.

An amount of chlorine dioxide of 0.3% (weight/weight) is added to thesethree suspensions.

The 3 suspensions are incubated for 2 hours 30 minutes at 75° C. Thepulp is then washed with 40 volumes of demineralized water and isrecovered in the form of a sheet.

The degree ISO of the three sheets obtained is measured.

The results are collated in Table 5.

TABLE 5 Amount of enzymes 10 0 0 employed in stage 2 in IU/g Amount ofchlorine 0.6 0.6 1.0 dioxide employed in stage 3 in % (weight/weight ofdry pulp) °ISO 88.5 85.5 87.9

This example shows that the xylanase according to the invention iseffective on eucalyptus pulp. Furthermore, it does not necessitate anypH adjustment, since it has the advantage of being active at the pH ofthe pulp, that is to say at a pH of approximately 9. This example showsthat the xylanase according to the invention is an alkaline xylanase.

This example also shows that, in comparison with what is obtainedwithout xylanase, the use of the xylanase according to the inventionbrings about an increase in brightness of at least 3° ISO for a fixedamount of ClO₂.

This example also shows that the use of the xylanase according to theinvention enables the amount of ClO₂ to be reduced by approximately 4 to5 kg/tonne of pulp, representing approximately 25 to 30% of the totalamount of ClO₂ needed.

EXAMPLE 14 Determination of the Nucleotide and Protein Sequence ofBacillus sp. 720/1 Xylanase

1. Extraction of Chromosomal DNA from Bacillus sp. Strain 720/1

From the culture B as obtained in Example 2, culturing of 200 ml ofBacillus sp. strain 720/1 is carried out in LB/C medium for 16 hours at37° C. LB/C medium is identical to the LBS/C medium described in Example1, without the addition of wheat bran.

When this culture has been prepared and is in stationary phase, it iscentrifuged (Beckman J 21, JA10 rotor) at 5,000 rpm for 10 minutes. Thecentrifugation pellet thereby obtained is taken up in 9 ml of Tris-HCl(tris(hydroxymethyl)aminomethane acidified with 0.1 M HCl) buffer at pH8, 0.1 M EDTA (ethylenediaminetetraacetic acid), 0.15 M NaCl containing18 mg of lysozyme; the suspension thereby obtained is incubated for 15minutes at 37° C.

The lysate thereby obtained is then treated with 200 μl of an RNAsesolution at a concentration of 10 mg/ml for 20 minutes at 50° C. 1 ml of10% (w/v) SDS (sodium dodecyl sulphate) solution is then added to thislysate. This lysate is then incubated for 30 minutes at 70° C.

The lysate is thereafter cooled to around 45° C., and 0.5 ml of asolution of proteinase K (sold by Boehringer Mannheim) at aconcentration of 20 mg/ml (prepared immediately before use) is thenadded to it.

The lysate is incubated at 45° C. with stirring until a transparentsolution is obtained.

Several phenol extractions are performed on this transparent solutionunder the conditions and following the procedures described in MolecularCloning—a laboratory manual—Sambrook, Fritsch, Maniatis—second edition,1989, on page E.3, until a clean interface is obtained, as describedtherein.

The DNA is precipitated with 20 ml of ethanol. The precipitate isrecovered by centrifugation at 5,000 rpm (Beckman J21, JA10 rotor) for 5minutes, and then suspended in 2 ml of TE buffer, pH 8.0, (10 mMTris-HCl, 1 mM EDTA, pH 8.0). This suspension contains the chromosomalDNA.

2. Construction of the Vector pUBR2002

The vector pUBR2002 (E. coli-Bacillus subtilis) was obtained fromplasmid pBR322 which is sold by the company Biolabs (ClontechLaboratories catalogue No. 6210-1) and the vector pUB131.

Two synthetic oligonucleotides are constructed by the techniquedescribed in Beaucage et al. (1981), Tetrahedron Letters, 22, pages1859-1882 and using β-cyanoethyl phosphoramidites in a Biosearch CycloneSynthesizer.

The sequences of these two oligonucleotides are as follows:

5′ - CCCCCCTACGTAGCGGCCGCCCCGGCCGGTAACCTAGGAAGTCAGCGCCCTGCACC - 3′ SEQID NO:14 and 5′ -CCCCCCTACGTAGGCCGGGGCGGCCGCGGTTACCTAGGGCCTCGTGATACGCCTAT - 3′ SEQ IDNO:15

These two oligonucleotides are used to perform a PCR amplification onplasmid pBR322 according to the technique described in MolecularCloning, a laboratory Manual—Sambrook et al., second edition, 1989,pages 14.18-14.19.

The PCR-amplified fragment contains the E. coli replicon limited on bothsides by the AvrII, BstEII, NotI, SfiI, SnabI restriction sites.

The approximately 2.8-kbp (kbp=1,000 base pairs) SnabI-SnabI fragment isligated with the vector pUB131 which has previously been subjected todigestion with SnabI. Construction of the vector pUB131 is described inExample 10 and in FIG. 7 of U.S. Pat. No. 5,352,603 (European PatentApplication 0,415,296), which is incorporated by reference.

The ligation technique is described by Sambrook et al. (pages1.68-1.69). All the ligations carried out in the examples in thisapplication were performed according to this technique.

The ligation thereby obtained is transformed into E. coli MC1061 cells[Clontech Laboratories, catalogue No. C-1070-1] by electroporation(Sambrook et al., pages 1.75-1.81). The transformed cells are culturedon Petri dishes containing LB agar medium, 100 μg/ml of ampicillin and10 μg/ml of kanamycin, at 37° C. for approximately 18 hours.

The plasmids are extracted from the colonies isolated by the alkalinelysis method (Sambrook et al., pages 1.25-1.28) and are subjected to arestriction analysis, the analysis described in Molecular Cloning, alaboratory Manual—Maniatis et al., 1982, Cold Spring Harbor Laboratory,pages 374-379.

A strain is obtained from which the vector which is designated pUBR2002(FIG. 3) is extracted.

3. Construction of a Bacillus sp. 720/1 Gene Library

From the suspension containing it, the chromosomal DNA is partiallycleaved with the restriction enzyme Sau3AI. The restriction conditionsare those described by Sambrook et al. (pages 5.28-5.32), except thatthese restriction conditions are increased by a factor of 10 in order toobtain a sufficient amount of DNA for the following purification steps.

The ratio of the amount of DNA employed to the amount of enzyme isadjusted in order to obtain a maxi mum of fragments between 4 and 7 kbp(kbp: 10³ base pairs) in size.

The set of fragments thereby obtained is then subjected to agarose (0.8%w/v) gel electrophoresis as described by Sambrook et al., pages6.01-6.19, and the fragments between 4 and 7 kbp in size are isolatedand purified by the method of filtration followed by centrifugationdescribed in Zhu et al., Bio/Technology 3, 1985, pages 1014-1016.

The DNA fragments thus purified are then ligated (according to themethod described by Sambrook et al., (pages 1.68-1.69) with plasmidpUBR2002 (E. coli-Bacillus subtilis) previously cut at the BamHI siteand dephosphorylated as described by Sambrook et al., (pages 1.60-1.61).

The ligation thereby obtained is used to transform E. coli MC1061 cellsby electroporation (Sambrook et al., pages 1.75-1.81).

4. Screening of the Gene Library

The transformed E. coli cells are cultured on Petri dishes containing LBagar medium, 0.8 g/l of AZCL-xylan and 100 μg/ml of ampicillin, forapproximately 24 hours at 37° C. A colony displaying a zone ofhydrolysis is isolated.

The plasmid present in this colony is extracted and isolated by thealkaline lysis technique described in Sambrook et al., pages 1.25-1.28.

A restriction analysis (Sambrook et al., page 1.85) is performed. Thisanalysis shows that the DNA fragment obtained, which contains thexylanase gene, is approximately 3.5 kbp (kbp=1,000 base pairs) in size.It is carried by the vector pUBR2002 which has been ligated.

The plasmid pUBR-720×1 (FIG. 4) is thereby obtained.

5. Cloning of a Chromosomal Fragment Containing the Xylanase Gene

Plasmid pUBR-720×1 is digested with restriction enzymes at the SwaI andSpeI sites. The xylanase gene is thereby obtained on an approximately1.5-kbp SwaI-SpeI DNA fragment.

This SwaI-SpeI DNA fragment is subjected to a treatment with the Klenowfragment of DNA polymerase (Sambrook et al., pages F.2-F.3).

The DNA preparation thereby obtained is ligated with the vector pUBR2002which has previously been digested with EcoRV and dephosphorylated.

The ligation is then transformed into E. coli MC1061 cells byelectroporation.

The transformed strains are selected on Petri dishes containing LB(Luria-Bertani) agar medium, 0.8 g/l of AZCL-xylan (Megazyme) and 100μg/ml of ampicillin, after growth at 37° C. for approximately 24 hours.

Colonies displaying a zone of hydrolysis are isolated. The plasmids areextracted from the colonies isolated by the alkaline lysis technique(Sambrook et al., pages 1.25-1.28), and are subjected to a restrictionanalysis (Maniatis et al., 1982, pages 374-379). This restrictionanalysis shows that the plasmid isolated, pUBR-720X11 (FIG. 5), containsthe xylanase gene on an approximately 1.5 kbp fragment of the Bacillussp. 720/1 chromosomal DNA.

Plasmid pUBR-720X11 is then used to transform E. coli JM109 cells(Clontech Laboratories catalogue No. C1005-1) by the CaCl₂ technique(Sambrook et. al., pages 1.82-1.84).

The transformed E. coli cells are cultured on Petri dishes containing LBagar medium, 0.8 g/l AZCL-xylan and 100 μg/ml of ampicillin. Aftergrowth at 37° C. for approximately 24 hours, a zone of hydrolysis isobserved around the colonies. This shows that the transformed E. colicells do indeed express the xylanase.

The technique enabling the DNA fragments to be dephosphorylated or thevectors to be linearized is described by Sambrook et al., (pages1.60-1.61).

A colony displaying a zone of hydrolysis is isolated. The plasmidpresent in this colony is extracted and isolated by the alkaline lysistechnique.

The sequence of the xylanase is established using the method describedin Sambrook et al. pages 13.15 and 13.17 (FIG. 13.3B), using plasmidpUBR-720X11 as template.

To initiate the sequence determination, synthetic oligonucleotides areprepared for hybridization with plasmid pUBR2002. The sequences of thesesynthetic ologonucleotides are as follows:

SEQ ID NO:16 5′ - ACGAGGAAAGATGCTGTTCTTGTAAATGAGT - 3′ and 5′ -TACCTTGTCTACAAACCCC - 3′ SEQ ID NO:17

The remainder of the sequence is determined by the use of othersynthetic oligonucleotides chosen on the basis of the newly determinedportions of the sequence.

The nucleotide sequence of the complete gene which codes for xylanase(SEQ ID NO:10) is thereby obtained (FIG. 2). The xylanase gene isobtained as an approximately 1.5-kbp fragment.

EXAMPLE 15 Construction of the Expression Vector pUBRD-720X11

The expression vector pUBRD-720X11 (FIG. 6) is a plasmid derived fromplasmid pUBR-720X11 from which the E. coli replicon has been removed.

The construction of plasmid pUBRD-720X11 from plasmid pUBR-720X11, asobtained in Example 14, is described below.

Plasmid pUBR-720X11 is digested with the restriction enzyme at the SnaBIsites for the purpose of removing the origin of replication of E. coli,according to the technique described in Example 14. An approximately5-kbp fragment is thereby obtained; it is ligated with itself accordingto the technique described in Example 14, to obtain plasmidpUBRD-720X11.

The ligation thereby obtained is used to transform competent Bacillussubtilis SE3 cells according to the technique described in DNA Cloning,vol. 11, ed. Glover, D. M., IRL Press Oxford, 1985, pages 9-11.

Bacillus subtilis strain SE3 was deposited on 21 Jun. 1993 at thecollection named Belgian Coordinated Collections of Microorganisms (LMGculture collection, Ghent, Belgium) in accordance with the BudapestTreaty under the number LMG P-14035.

The transformed cells are cultured on Petri dishes containing LB(Luria-Bertani) agar medium, 0.8 g/l of AZCL-xylan and 25 μg/ml ofkanamycin, at 37° C. for approximately 18 hours. After growth, coloniesdisplaying a zone of hydrolysis are isolated.

The isolated colonies are subjected to a plasmid analysis by enzymerestriction for the purpose of verifying that the construction of theplasmid is correct, according to the technique described in Example 14.

A strain is obtained from which the vector which is designatedpUBRD-720X11 may be isolated.

EXAMPLE 16 Transformation of Bacillus licheniformis Strain SE2 delap6with the Expression Vector pUBRD-720X11

Plasmid pUBRD-720X11 described in Example 15 is extracted from its host,isolated and purified (Sambrook et al., 1989, p. 1.25-1.28).

A culture of Bacillus licheniformis strain SE2 delap6 is prepared.Bacillus licheniformis strain SE2 delap6 and the culturing thereof aredescribed in Examples 27 and 28 of European Patent Application0,634,490, which is incorporated by reference.

Bacillus licheniformis strain SE2 delap6 was prepared from Bacilluslicheniformis strain SE2, which was deposited on 21 Jun. 1993 at thecollection named Belgian Coordinated Collections of Microorganisms (LMGculture collection, Ghent, Belgium) in accordance with the BudapestTreaty under the number LMG P-14034.

Bacillus licheniformis strain SE2 delap6 is then transformed withplasmid pUBRD-720X11 according to the protoplast technique (Maniatis etal., p. 150-151).

The transformed strain [Bacillus licheniformis SE2 delap6(pUBRD-720X11)] is selected on Petri dishes containing LB agar medium,0.8 g/l of AZCL-xylan and 25 μg/ml of kanamycin. It is then isolated andpurified by screening, that is to say by being applied and streaked toobtain single colonies at the surface of LB (Luria-Bertani) agar mediumwhich is described in Molecular Cloning-Laboratory Manual (Sambrook etal.), 1989, p. A.4.

EXAMPLE 17 Production of Xylanase by B. licheniformis SE2 delap6(pUBRD-720X11)

B. licheniformis strain SE2 delap6 transformed by plasmid pUBRD-720X11,as obtained in Example 16, is cultured for 17 hours at 37° C. in an LBculture medium supplemented with 0.5% (w/v) of glucose and 20 μg/ml ofkanamycin.

This culture is transferred (5% v/v) to 50 ml of M2 medium supplementedwith 20 μg/ml of kanamycin.

M2 medium contains 30 g of soya flour, 75 g of soluble starch, 2 g ofsodium sulphate, 5 mg of magnesium chloride, 3 g of NaH₂PO₄, 0.2 g ofCaCl₂.H₂O and 1,000 ml of water. The pH of this M2 medium is adjusted to5.8 with 10 N NaOH before it is sterilized.

The culture is incubated with orbital shaking at the rate of 250 rpmwith an amplitude of approximately 2.54 cm for 80 hours at 37° C. After80 hours, the biomass is removed by centrifugation (Beckman J21, JA10rotor) at 5,000 rpm for 10 minutes. The centrifugation supernatant isretained. The enzyme activity is measured on this supernatant accordingto the technique described in Example 2, and the presence of an xylanaseactivity is noted.

EXAMPLE 18 Construction of the Vector pUBC2001

The vector pUBC2001 (E. coli-Bacillus) (FIG. 7) is a plasmid derivedfrom the plasmid pUBC131 containing, as sole difference, the presence oftwo additional restriction sites: BstEII and PacI. Construction of thevector UBC131 is described in Example 11 and in FIG. 8 of U.S. Pat. No.5,352,603 (European Patent Application 0,415,296), which is incorporatedby reference.

The construction of this plasmid is described below.

Four synthetic oligonucleotides are constructed according to thetechnique described in Example 14.

The sequences of these four oligonucleotides are as follows:

5′ - CGGTCGCCGCATACACTA - 3′ SEQ ID NO:18 5′ -CCCCCCCCCGGTAACCTGCATTAATGAATCGGCCAA - 3′ SEQ ID NO:19 5′ -CCCCCCCCCGGTTACCGTATTTATTAACTTCTCCTAGTA - 3′ SEQ ID NO:20 5′ -CCCCCCTCTAGATTAATTAACCAAGCTTGGGATCCGTCGACCTGCAGATC - 3′ SEQ ID NO:21

The two oligonucleotides having the sequences SEQ ID NO: 18 and 19 areused to perform a PCR amplification on the vector pUBC131 according tothe PCR technique described in Example 14. The PCR-amplified fragment,containing a portion of the ampicillin resistant gene and the functionsneeded for replication in E. coli, is subjected to a restriction withScaI and BstEII, generating an approximately 1.5-1.6-kbp fragment.

A second PCR amplification is carried out on the vector pUBC131, usingthe oligonucleotides having the sequences SEQ ID NO: 20 and 21 andaccording to the technique described in Example 14. The PCR-amplifiedfragment contains a portion of the vector pUBC131. This fragment issubjected to a restriction with BstEII and EcoRI, generating anapproximately 1.4-1.5-kbp fragment.

The two fragments thereby obtained are ligated together, according tothe technique described in Example 14, with the vector pUBC131 which haspreviously been subjected to a double digestion with EcoRI and ScaI,according to the technique described in Example 14.

The ligation thereby obtained is used to transform E. coli MC1061 cellsby electroporation according to the technique described in Example 14.The transformed cells are cultured on Petri dishes containing LB agarmedium, 100 μg/ml of ampicillin and 10 μg/ml of kanamycin, at 37° C. forapproximately 18 hours.

After growth, the isolated colonies are subjected to a plasmid analysisby enzyme restriction according to the technique described in Example14.

A strain is obtained from which the vector which is designated pUBC2001may be extracted.

EXAMPLE 19 Construction of the Expression Vector pC-BPX-PRE-2003

The expression vector pC-BPX-PRE-2003 (E. coli-Bacillus) (FIG. 8) is anexpression vector derived from plasmid pUBC2001. It contains thepromoter derived from the gene which codes for Bacillus pumilus PRL B12xylanase and the presequence which codes for the signal peptide ofBacillus pumilus PRL B12 xylanase. The method for preparing andobtaining the promoter derived from the gene which codes for Bacilluspumilus PRL B12 xylanase and the presequence which codes for the signalpeptide of Bacillus pumilus PRL B12 xylanase is described in Example 17and in FIG. 1 of European Patent Application 0,634,490, which isincorporated in this application by reference.

The sequence of the promoter derived from the gene which codes forBacillus pumilus PRL B12 xylanase is described in the presentapplication under the number SEQ ID NO: 26. The sequence of thepresequence which codes for the signal peptide of Bacillus pumilus PRLB12 xylanase is described in the present application under the numberSEQ ID NO: 27.

The construction of plasmid pC-BPX-PRE-2003 is described below.

Two synthetic oligonucleotides are constructed according to thetechnique described in Example 14.

The sequences of these two oligonucleotides are as follows:

SEQ ID NO:22 5′ - CCCCCCTGAAATCAGCTGGACTAAAAGGGATGCAATTTC - 3′ SEQ IDNO:23 5′ - CCCCCCGTCGACCGCATGCGCCGGCACAGC - 3′

These two oligonucleotides are used to perform a PCR amplification onthe plasmid pUB-BPX12 according to the technique described in Example14. Construction of the plasmid pUB-BPX12 is described in Example 17 andin FIG. 4 of European Patent Application 0,634,490, which isincorporated by reference.

The use of the oligonucleotide having the sequence SEQ ID NO: 22 makesit possible, by changing one nucleotide, to remove, upstream of the B.pumilus PRL B12 xylanase promoter, the SphI restriction site, located atapproximately 5.5 kbp, normally present in pUBC2001. The change ofnucleotide is represented by the nucleotide underlined in the sequenceSEQ ID NO: 22 above, and relates to the replacement of C by G for theSphI site (SphI=GCATGC).

The sequence SEQ ID NO: 23 enables a new SphI site to be created at theend of the presequence which codes for the signal peptide of B. pumilusPRL B12 xylanase, by changing only one nucleotide of the Ala codon [25],that is to say by changing GCG to GCT.

The PCR amplified fragment, containing the promoter derived from thegene which codes for B. pumilus PRL B12 xylanase and the presequencewhich codes for the signal peptide of B. pumilus PRL B12 xylanase, issubjected to a restriction with PvuII and SphI, generating anapproximately 0.7-kbp fragment, according to the technique described inExample 14.

The approximately 0.7-kbp PvuII-SphI fragment is ligated with the vectorpUBC2001 which has previously been subjected to a double digestion withPvuII and SphI, according to the techniques described in Example 14.

The ligation thereby obtained is used to transform E. coli MC1061 cellsby electroporation according to the technique described in Example 14.The transformed cells are cultured on Petri dishes containing LB agarmedium, 100 μg/ml of ampicillin, at 37° C. for approximately 18 hours.

After growth, the isolated colonies are subjected to a plasmid analysisby enzyme restriction according to the technique described in Example14.

A strain is obtained from which the vector which is designatedpC-BPX-PRE-2003 may be extracted.

EXAMPLE 20 Construction of the Expression Vector pC-BPX-PRE-720X

The expression vector pC-BPX-PRE-720X (E. coli-Bacillus) (FIG. 9) is anexpression vector containing the promoter derived from the gene whichcodes for B. pumilus PRL B12 xylanase and the presequence which codesfor the signal peptide of B. pumilus PRL B12 xylanase and the sequenceof the gene which codes for the mature portion of Bacillus sp. 720/1xylanase.

The expression vector pC-BPX-PRE-720X contains the sequence SEQ ID NO:1, the nucleotide sequence of the gene which codes for the matureportion of Bacillus sp. 720/1 xylanase.

The construction of plasmid pC-BPX-PRE-720X is described below.

Two synthetic oligonucleotides are constructed according to thetechnique described in Example 14.

The sequences of these two oligonucleotides are as follows:

SEQ ID NO:24 5′ - CCCCCCGCATGCGCAAATCGTCACCGACAATTCCATTGG - 3′ 5′ -TACCTTGTCTACAAACCCC - 3′ SEQ ID NO:25

These two oligonucleotides are used to perform a PCR amplification onplasmid pUBR-720X11, as obtained in Example 14, and according to thetechnique described in Example 14.

The PCR amplified fragment containing the sequence of gene which codesfor the mature portion of Bacillus sp. 720/1 xylanase is subjected to arestriction with SphI and SacI, generating an approximately 0.8-kbpfragment, according to the technique described in Example 14.

The SphI-SacI fragment is ligated with the vector pC-BPX-PRE-2003 whichhas previously been subjected to a double digestion with SphI and SacI,according to techniques described in Example 14. Ligation at the SphIrestriction site enables a translational fusion of the signal sequenceof the gene which codes for B. pumilus PRL B12 xylanase with thesequence of the gene which codes for the mature portion of Bacillus sp.720/1 xylanase to be created.

The ligation thereby obtained is used to transform E. coli MC1061 cellsby electroporation according to the technique described in Example 14.The transformed cells are cultured on Petri dishes containing LB agarmedium, 0.8 g/l of AZCL-xylan and 100 μg/ml of ampicillin, at 37° C. forapproximately 18 hours. Colonies displaying a zone of hydrolysis areisolated.

After growth, the isolated colonies are subjected to a plasmid analysisby enzyme restriction according to the technique described in Example14.

A strain is obtained from which the vector which is designatedpC-BPX-PRE-720X may be extracted.

EXAMPLE 21 Construction of the Vector pBPXD-PRE-720X

The vector pBPXD-PRE-720X (Bacillus) (FIG. 10) is an expression vectorderived from plasmid pUB131. It contains the promoter derived from thegene which codes for B. pumilus PRL B12 xylanase and the presequencewhich codes for the signal peptide of B. pumilus PRL B12 xylanase andthe sequence of the gene which codes for the mature portion of Bacillussp. 720/1 xylanase.

The construction of plasmid pBPXD-PRE-720X is described below.

Plasmid pC-BPX-PRE-720X obtained in Example 20 is subjected to arestriction with PvuII and EcoRI, generating an approximately 1.5-kbpfragment, according to the technique described in Example 14.

The approximately 1.5-kbp fragment is ligated with the vector pUB131which has previously been subjected to a double digestion with PvuII andEcoRI, according to techniques described in Example 14.

The ligation thereby obtained is used to transform B. subtilis SE3 cellsaccording to the electroporation technique described in Example 14. Thetransformed cells are cultured on Petri dishes containing LB agarmedium, 0.8 g/l of AZCL-xylan and 25 μg/ml of kanamycin, at 37° C. forapproximately 18 hours. Colonies displaying a broad zone of hydrolysisare isolated.

After growth, the isolated colonies are subjected to a plasmid analysisby enzyme restriction according to the technique described in Example14.

A strain is thereby obtained from which the vector which is designatedpBPXD-PRE-720X may be extracted.

EXAMPLE 22 Transformation of Bacillus licheniformis SE2 delap6 with theExpression Vector pBPXD-PRE-720X

Plasmid pBPXD-PRE-720X (FIG. 10) described in Example 21 is extractedfrom its host, isolated and purified.

A culture of B. licheniformis strain SE2 delap6 is prepared according tothe technique described in Example 16. This strain is then transformedwith this plasmid according to the protoplast technique described inExample 16.

The transformed strain [B. licheniformis SE2 delap6 (pBPXD-PRE-720X)] isselected from Petri dishes containing LB agar medium, 0.8 g/l ofAZCL-xylan and 25 μg/ml of kanamycin. The strain is isolated andpurified by screening according to the technique described in Example16.

EXAMPLE 23 Production of Xylanase by B. licheniformis SE2 delap6(pBPXD-PRE-720X)

An assay is performed which is identical to the one described in Example17, but with B. licheniformis strain SE2 delap6 transformed by plasmidpBPXD-PRE-720× as obtained in Example 22.

The enzyme activity is measured according to the technique described inExample 2 on the supernatant obtained, and the presence of a xylanaseactivity is noted.

EXAMPLE 24 Preparation and Isolation of the Xylanase Produced by B.licheniformis Strain SE2 delap6 (pBPXD-PRE-720X)

The xylanase produced by B. licheniformis strain SE2 delap6 transformedby plasmid pBPXD-PRE-720X, as obtained in Example 22, is isolated andpurified. This strain is cultured according to the protocol described inExample 23.

The xylanase obtained is isolated and purified according to the protocoldescribed in Example 3 of European Patent Application 0,634,490, whichis incorporated in this application by reference.

EXAMPLE 25 Amino Acid Sequence of the Xylanase Produced by B.licheniformis strain SE2 delap6 (pBPXD-PRE-720X)

The sequence of the first 50 amino acids of the xylanase produced by B.licheniformis strain SE2 delap6 (pBPXD-PRE-720X) is determined using asequencing apparatus (HP G1000A, Hewlett-Packard) and according to theinstruction leaflet of this apparatus.

The xylanase isolated and purified as described in Example 24 is used.

It is verified that the sequence obtained is identical to the onedescribed in Example 4 for the xylanase produced by Bacillus sp. strain720/1.

EXAMPLE 26 Determination of the Molecular Weight of the XylanaseProduced by B. licheniformis strain SE2 delap6 (pBPXD-PRE-720X)

The molecular weight of the xylanase produced by B. licheniformis strainSE2 delap6 (pBPXD-PRE-720X) is determined according to the protocoldescribed in Example 7 and employing the xylanase isolated and purifiedas described in Example 24.

Staining with Coomassie blue reveals a polypeptide of molecular weightbetween 25 and 26 kD, which is identical to that of the xylanaseproduced by Bacillus sp. strain 720/1.

1. An isolated DNA molecule comprising the nucleotide sequence of SEQ IDNO:4 which codes for a Bacillus sp. 720/1 xylanase.
 2. The isolated DNAmolecule according to claim 1, wherein said DNA molecule comprises apromoter comprising SEQ ID NO: 26 and a presequence comprising SEQ IDNO:
 27. 3. An expression vector or chromosomal integration vectorcontaining the DNA molecule according to claim 1 or
 2. 4. A transformedBacillus strain comprising the DNA molecule according to claim 1 or 2.5. The transformed strain according to claim 4, wherein said strain is aBacillus licheniformis or Bacillus pumilus strain.